Abstract

Most cases of CLL express BCL2 constitutively at levels comparable to those seen in follicular lymphoma with t(14;18)(q32;q21) and BCL2 is considered to play a pivotal role in the suppression of apoptosis in this disease. The BCL2 antagonist ABT-263 has recently entered phase I/IIa clinical trials for potential treatment of CLL. ABT-263 and ABT-737 are rationally designed BH3 mimetics that are thought to act by displacing pro-apoptotic BIM from BCL2 and thus activating the apoptotic cascade. While being resistant towards some commonly used apoptotic stimuli like etoposide or TRAIL, we found that all primary CLL cells are highly and rapidly sensitive to ABT-737, with an average EC50 in 60 patients of only 6.7 nM and after only 4 h of treatment. ABT-737 induced BAX/BAK oligomerization followed by rapid and extensive cytochrome c release, caspase activation and chromatin condensation. Incubating unfractionated samples in a whole blood assay did not significantly alter sensitivity. Notably, sensitivity of CLL cells to ABT-737 was independent of the patient’s stage of disease, p53 mutation or function, cytogenetics, or IGVH mutational status. Furthermore, ABT-737-induced apoptosis was observed irrespective of previous treatment or in vivo resistance to fludarabine, suggesting that ABT-737 might induce apoptosis even in patients with highly progressive and otherwise resistant disease. We also analyzed expression levels of BCL2 and MCL1 in CLL patients by western blot, since MCL1 is considered to be the most important factor for resistance towards ABT-737. Interestingly, sensitivity to ABT-737 was independent of BCL2 and MCL1 levels in CLL, indicating that expression of MCL1 was not sufficient to render CLL resistant to ABT-737. In addition, we further induced expression of MCL1 by either IL4 or interferon γ treatment, which resulted only in a minor delay of apoptosis that was easily overcome by treatment with slightly higher concentrations of ABT-737. Hence, our data suggest that in contrast to previous reports obtained in a variety of cell lines, MCL1 upregulation is not sufficient to induce resistance to ABT-737 in primary CLL cells. Next, we investigated whether exposure to ABT-737 resulted in a displacement of BH3-only proteins from BCL2. Surprisingly, we could not detect a significant displacement of BIM from BCL2. In contrast, we found that the multidomain proapoptotic protein BAK is directly bound to BCL2 in CLL and this interaction is abrogated by ABT-737, indicating that ABT-737 can directly displace BAK but not BIM from BCL2. Taken together, our results indicate that targeting BCL2 with ABT-263 or ABT-737 may be a highly promising strategy for treatment of CLL.

Author notes

Disclosure: No relevant conflicts of interest to declare.